Regulation Of Hematopoietic Stem Cells Research Articles

The Role of Stathmin-1 in the Regulation of Hematopoietic Stem Cells

Stathmin-1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of alpha-beta tubulin heterodimers. Also known as Oncoprotein 18 (OP18), Stathmin-1 is highly expressed in a wide variety of tumors, including high-risk myelodysplastic syndromes and acute leukemias (Machado-Neto et al, 2014). Stathmin-1 is also highly expressed in normal hematopoietic stem cells (HSCs), and its loss is associated with impaired erythroid differentiation and altered megakaryopoiesis (Ramlogan-Steel et al, 2021). While these prior studies have established a role of Stathmin-1 in normal erythro-megakaryopoiesis, its role in hematopoiesis- particularly in hematopoietic stem and progenitor cells- is not clear. Given the high expression of Stathmin-1 in HSCs and the importance of microtubule dynamics to HSC differentiation, we hypothesized that Stathmin-1 loss may impact normal HSC function. To address the role of Stathmin-1 in HSCs, we characterized HSC phenotype and function in a Stathmin-1 knockout mouse (Stmn1-/-). Under homeostatic conditions, Stmn1-/- HSC number and frequency are normal compared to wild-type (WT) mice. However, Stmn1-/- HSCs have markedly impaired function, as their bone marrow (BM) failed to engraft in competitive transplants (10% vs. 60% chimerism at 24 weeks post-transplant p<0.0001). Competitive intra-tibia transplants were then done to circumvent potential homing defects, and again Stmn1-/- BM displayed a significant engraftment defect compared to WT (10% vs. 78% chimerism at 24 weeks p<0.0001). Of note, Stmn-1-/- HSCs performed equivalently to WT HSCs in an in vivo homing assay, further suggesting their lack of engraftment is not due to homing defects. Stmn1-/- BM cells were capable of engraftment in non-competitive transplants, albeit less robustly than WT cells (42% vs. 90% p<0.01). Treatment of WT and Stmn1-/- mice with one single dose of the chemotherapeutic agent 5-fluorouracil showed that Stmn1-/-mice have slower tri-lineage recovery than their WT littermates (p<0.01 at day 15). Consistent with their reduced in vivo function, Stmn1-/- HSCs produced fewer colonies than WT HSCs when plated in Methocult. They were also not capable of serial replating, supporting that loss of Stathmin-1 is associated with impaired HSC function. Consistent with its known role in regulating microtubules, Stmn1-/- HSCs displayed altered microtubule morphology by confocal microscopy with a lack of normal nuclear invaginations compared to WT HSCs. Analysis of cycling status revealed that Stmn1-/- HSCs are more quiescent than WT HSCs at baseline (1.3% vs. 3.4% at S/G2/M phase) and in response to 24 hours of G-CSF stimulation (3.4% vs. 7% at S/G2/M phase). In addition, Stmn1-/- HSCs have higher rates of apoptosis than their WT counterparts (18.5% vs. 9.90%), suggesting that Sthamin-1 loss leads to both impaired HSC cycling and increased cell death. We next performed RNA-seq on sorted Stmn1-/- and WT HSCs to gain further mechanistic insight into their impaired function. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function, and cell differentiation in the Stmn1-/- HSCs compared to WT controls. Consistent with these results, measurement of mitochondrial function (oxygen consumption rate) using the Seahorse analyzer showed that Stmn1-/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT. Together, these data suggest a model whereby Stathmin-1 regulates microtubule dynamics in HSCs to promote optimal mitochondrial function to support cellular cycling, survival and self-renewal.

Ing4-Deficiency Confers Resistance to Inflammatory Stress through Enhanced Hematopoietic Stem Cell Quiescence

In a screen of epigenetic regulators of hematopoiesis, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in hematopoietic stem and progenitor cell specification in embryonic zebrafish. To further our understanding of how Ing4 regulates adult hematopoiesis, we have extended these studies to examine murine hematopoiesis in the absence of Ing4. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 promotes stem cell-like characteristics in malignant cells and is a frequent target of inactivation in various types of cancer. Studies in cancer cell lines have shown that mutations in Ing4 cause deregulation of NF-kB target gene expression and upregulation of c-Myc target gene expression, making it a fascinating candidate for regulation of hematopoiesis. In our mouse model of Ing4 deficiency, we have examined murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis, with a 2% increase in long-term hematopoietic stem cells (LT-HSCs) and a 12% increase in short-term HSCs (ST-HSCs). Highly elevated cytokine expression is also observed in bone marrow cells. Using bulk RNA-sequencing of HSCs, we found that Ing4-deficient HSCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Despite these characteristics of activation, Ing4 deficiency induces a G0 arrest in HSCs. LT-HSCs show a 15% increase in cells arrested in G0, while ST-HSCs show an increase of 11%. Both populations of HSCs also contain significantly lower levels of reactive oxygen species. Taken together, these data suggest that Ing4-deficient HSCs are in a unique primed state, where they are quiescent, but express elevated levels of genes associated with activation. To test the function of Ing4-deficient HSCs, we examined hematopoiesis following low-dose irradiation (LDI). Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multi-lineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. Exposure to inflammatory cytokines typically impairs HSC function in transplantation and renders them incapable of contributing to multi-lineage chimerism, making our results quite striking. We sought to target the dysregulated pathways in Ing4-deficient HSPCs to rescue the effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the dysregulated pathways observed in Ing4-/- HSCs, and Ing4 has been reported to negatively regulate c- Myc activity directly in cancer cell line models. Wild-type and Ing4-deficient mice were subjected to LDI, then mock or drug-treated with the c-Myc inhibitor, 10058-F4, for 7 days. Ing4-deficient HSCs are triggered into cycling upon treatment, with a 15% decrease in cells in G0. ST-HSCs show a 5% reduction of cells in G0. These data suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoietic stem cells. Our work also provides key tools for understanding how HSCs may overcome inflammatory stress by providing a model for robust HSC function under stress.

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.

Rker-050, a Novel Activin Receptor Type II Ligand Trap, Rescued Anemia and Reduced Bone Loss in a Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and dysplasias resulting from molecular defects and dysregulated signaling that govern the regulation of hematopoietic stem and progenitor cells (HSPCs). Crosstalk between cells in the osteo-hematopoietic (O-H) niche are necessary for the maintenance and self-renewal of HSPCs. Additionally, MDS patients commonly present with osteopenia and osteoporosis further demonstrating the critical interplay between the O-H cells in the bone marrow (BM). Alterations in the interaction between bone precursors and HSPCs within the BM microenvironment have been linked to the pathophysiology of MDS. Transforming growth factor-beta (TGF-β) superfamily signaling is instrumental in regulating both normal hematopoiesis and bone homeostasis. KER-050 is a modified investigational activin receptor type II ligand trap designed to bind and inhibit select TGF-β superfamily ligands, including activin A, activin B, GDF8 and GDF11, to promote hematopoiesis and bone growth. Preclinical studies have demonstrated that treatment with a research form of KER-050 (RKER-050) increased bone mass and multi-lineage hematopoiesis in healthy mice. To further investigate the effect of RKER-050 on hematopoietic and bone systems within the MDS disease setting, the NUP98/HOX13 MDS mouse model was utilized. Five-month-old male MDS mice were treated with vehicle (VEH, n=10), or RKER-050 (050, 7.5mg/kg, n=9) once weekly for 6 weeks along with a wildtype vehicle (WT, n=7) control group. To assess changes in hematological parameters, CBCs were measured, bone marrow (BM) and splenocytes were characterized via flow cytometry. To investigate alterations of bone microarchitecture, femurs were assessed using microCT imaging. MDS-VEH mice showed a reduction in red blood cells (RBC; 30%), hemoglobin (HGB; 19%), hematocrit (HCT; 13%) and reticulocytes (Retic; 16%) compared to WT; all hallmarks of human MDS. Relative to MDS-VEH, MDS-050 showed increased RBC, HGB, HCT and Retic by 25%, 13%, 10% and 39%, respectively. The erythroid Ter119 positive (Ter119+) cell population was slightly reduced in BM and spleen of MDS-VEH mice compared to WT. The BM Ter119+ cells were unchanged in the MDS-050 cohort relative to MDS-VEH; however, the spleen Ter119+ cells were increased similar to the levels in WT mice. Specific erythroid precursor populations were increased in BM and spleen of MDS-VEH mice versus WT, consistent with the ineffective erythropoiesis in this model. Interestingly, erythroid precursor populations were similar between MDS-VEH and MDS-050, yet MDS-050 exhibited recovered RBC production, suggesting an increase in maturation of erythroid precursors with RKER-050 treatment. Given the importance of the O-H niche in regulating hematopoiesis along with osteoporosis and osteopenia being comorbidities of MDS, bone microarchitecture was also analyzed. In the distal femoral trabecular bone, relative to WT mice, MDS-VEH mice had significant increases in trabecular (Tb) separation (33%) and decreases in Tb number (28%), changes that are consistent with reduced bone strength. Bone volume (34%) and bone volume fraction (26%) decreased but were not statistically significant. MDS-050 treated mice had a significant increase in bone volume (71%), higher bone volume fraction (88%), increased Tb number (44%) and decreased Tb spacing (20%) compared to the MDS-VEH, supporting RKER-050's role in improving bone mass. Taken together, these results suggest that RKER-050 can mitigate anemia and bone loss in an MDS mouse model, potentially by rebalancing hematopoiesis and bone turnover. In particular, the ability of RKER-050 to improve bone microarchitecture within the trabecular region, where the O-H niche resides, could be an important step in reestablishing healthy blood cell production. Furthermore, exploratory PD data from an ongoing phase 2 clinical trial demonstrate improved blood parameters with KER-050 treatment, including platelet and RBC in MDS patients (Tan et al. 2022 EHA #P776), and changes in bone biomarkers are currently being evaluated in the study. KER-050, thus, represents a potentially promising approach for patients with MDS and other hematological diseases, including myelofibrosis, where ineffective hematopoiesis and defects in bone homeostasis occur.

Micrornas Analysis in Patients with Myelodysplastic Syndrome. Possible Implications in Risk Stratification

Background. MicroRNAs (miRNAs) are significant regulators of human hematopoietic stem cells (HSC), and their deregulation contributes to hematological malignancies. Systematic analysis of miRNA expression and function during hematopoiesis has revealed regulatory loops where miRNAs contribute to hematopoietic differentiation and function. Among the identified miRNAs, miR-155 can regulate myelopoiesis and erythropoiesis and its overexpression along with the overexpression of miR-125 and miR-181 has been suggested to have functional effects in the regulation of HSC homeostasis. Myelodysplastic syndromes (MDS) are characterized by dysfunctional HSC, ineffective blood cell production, progressive marrow failure, and exhibit a significant risk of progression to acute myeloid leukemia (AML). Although the pathogenesis of MDS has not been fully understood, various alterations of microRNAs (miRNAs) have been reported in MDS. There is recent evidence that the miRNAs mediated post-transcriptional control of gene expression is key component in the pathogenesis of MDS and AML. Several studies have reported that differentially expressed microRNAs are associated with the transformation of MDS to AML and clinical outcomes. Aim. In this study we examined the potential prognostic impact of six previously described miRNAS (miR-181a-2-3p, miR-124-3p, miR-550a-3p, miR-155-5p, miR-151a-3p, and miR-125b-5p) on the pathogenesis of MDS. Methods. Total RNA was extracted from diagnostic bone marrow samples of 41 MDS patients. Patients were stratified per the revised International Prognostic Scoring System (IPSS-R). After polyadenylation of 1 μg total RNA by poly(A) polymerase and subsequent reverse transcription with an oligo-dT adapter primer, miRNAs transcript levels were determined using a reverse transcription quantitative real-time PCR method, based on SYBR Green chemistry. U6 was used as reference gene. Relative microRNA transcript levels were calculated by the 2-ΔΔct method. The IBM SPSS statistics, version 26 (IBM Corporation, North Castle, NY, USA) was used for the statistical analysis of the results. Results. The basic characteristics of the patients are shown in Table 1. Of the microRNAs analyzed in this study, miR-125b-5p, miR-155-5p, miR-181a-2-3p transcript levels were significantly elevated in higher (intermediate, high, and very high) risk MDS than in lower (very low and low) risk MDS (p=0.012, p=0.010, and p=0.024 respectively). MiR-125b-5p, miR-151a-3p, miR-155-5p, miR-181a-2-3p, and miR-550a-3p presented a positive correlation with the percentage of bone marrow blasts [p(0.001-0.05)], while a negative correlation was observed between the expression levels of mir-151-a-3p, miR181a-2-3p, miR-550a-3p, and the neutrophil count (p=0.05, 0.013, 0.010, and 0.007 respectively). None of the remaining factors that were tested (MDS type per the 2016 WHO classification, gender, age at diagnosis, hemoglobin levels, platelet count, and cytopenias) were correlated with the miRNAs levels. Discussion. In this study we demonstrated the notable difference in the pattern of miRNA expression between lower and higher risk MDS patients. It has been shown that deregulation of miR-155-5p and miR-181a-2-3p may promote aberrant hematopoietic stem cell proliferation, differentiation, and progression to AML. Moreover, mice transplanted with miR-155-5p transfected stem cells developed a myeloproliferative disorder with abnormal granulocyte morphology analogous to MDS, suggesting a role in the higher risk MDS phenotype. Finally, it has been reported that miR-181a-3p was strongly expressed in the cytoplasm of high risk MDS cells, in contrast to low risk samples which displayed reduced levels. The identification of miRNAs that distinguish lower risk from higher risk MDS indicates that microRNA expression profile may be exploited to assist with making treatment decisions. Given that certain miRNAs can discriminate MDS subtypes and risk groups, it is not unlikely to think that an miRNA signature could be a useful tool for MDS risk stratification. Based on our current findings and other previously reported results, we continue the study in a larger cohort of patients. Targeting miRNAs may provide new treatment options for MDS Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of hematopoietic and leukemia stem cells by regulatory T cells.

Adult bone marrow (BM) hematopoietic stem cells (HSCs) are maintained in a quiescent state and sustain the continuous production of all types of blood cells. HSCs reside in a specialized microenvironment the so-called HSC niche, which equally promotes HSC self-renewal and differentiation to ensure the integrity of the HSC pool throughout life and to replenish hematopoietic cells after acute injury, infection or anemia. The processes of HSC self-renewal and differentiation are tightly controlled and are in great part regulated through cellular interactions with classical (e.g. mesenchymal stromal cells) and non-classical niche cells (e.g. immune cells). In myeloid leukemia, some of these regulatory mechanisms that evolved to maintain HSCs, to protect them from exhaustion and immune destruction and to minimize the risk of malignant transformation are hijacked/disrupted by leukemia stem cells (LSCs), the malignant counterpart of HSCs, to promote disease progression as well as resistance to therapy and immune control. CD4+ regulatory T cells (Tregs) are substantially enriched in the BM compared to other secondary lymphoid organs and are crucially involved in the establishment of an immune privileged niche to maintain HSC quiescence and to protect HSC integrity. In leukemia, Tregs frequencies in the BM even increase. Studies in mice and humans identified the accumulation of Tregs as a major immune-regulatory mechanism. As cure of leukemia implies the elimination of LSCs, the understanding of these immune-regulatory processes may be of particular importance for the development of future treatments of leukemia as targeting major immune escape mechanisms which revolutionized the treatment of solid tumors such as the blockade of the inhibitory checkpoint receptor programmed cell death protein 1 (PD-1) seems less efficacious in the treatment of leukemia. This review will summarize recent findings on the mechanisms by which Tregs regulate stem cells and adaptive immune cells in the BM during homeostasis and in leukemia.

Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function

SummaryInteraction with microenvironmental factors is crucial for the regulation of hematopoietic stem cell (HSC) function. Stroma derived factor (SDF)-1α supports HSCs in the quiescent state and is central to the homing of transplanted HSCs. Here, we show that integrin signaling regulates Sdf-1α expression transcriptionally. Systemic deletion of Periostin, an Integrin-αv ligand, showed increased expression of Sdf-1α in bone marrow (BM) niche. Pharmacological inhibition or CRISPR-Cas9-mediated deletion of SRC, resulted in a similar increase in the chemokine expression in vitro. Importantly, systemic SRC-inhibition led to increase in SDF-1α levels in BM plasma. This resulted in a robust increase (14.05 ± 1.22% to 29.11 ± 0.69%) in the homing efficiency of transplanted HSCs. In addition, we observed enhancement in the recovery of blood cell counts following radiation injury, indicating an enhanced hematopoietic function. These results establish a role of SRC-mediated integrin signaling in the transcriptional regulation of Sdf-1α. This mechanism could be harnessed further to improve the hematopoietic function.

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Zeb1 sustains hematopoietic stem cell functions by suppressing mitofusin-2-mediated mitochondrial fusion

Metabolic status is essential in maintaining normal functions of hematopoietic stem cells (HSCs). However, how the dynamic of the mitochondrion, as a central organelle in metabolism, is molecularly regulated to orchestrate metabolism and HSC stemness remains to be elucidated. Here, we focus on the role of Zeb1, a well-characterized epithelial-to-mesenchymal transition (EMT) inducer which has been demonstrated to confer stem-cell-like characteristics in multiple cancer types in stemness regulation of HSCs. Using a Zeb1-tdTomato reporter mouse model, we find that Zeb1+Lin−Sca-1+c-Kit+ cells (Zeb1+-LSKs) represent a subset of functional long-term HSCs. Zeb1+LSKs exhibit a reduced reactive oxygen species (ROS) level, low mitochondrial mass, low mitochondrial membrane potential (MMP), and particularly small, round fragmented mitochondria. Of note, ectopic expression of Zeb1 leads to a fragmented mitochondrial morphology with a low mitochondrial metabolic status in EML cells. In addition, Zeb1-knockout (Zeb1-KO) LSKs from fetal liver display an exhausted stem-cell activity. Zeb1 deficiency results in elongated and tubulated mitochondria with increased mitochondrial mass, elevated MMP, and higher ROS production. Mechanistically, Zeb1 acts as a transcriptional suppressor on the key mitochondrial-fusion protein Mitofusin-2 (encoded by Mfn2). We highlight an important role of Zeb1 in the regulation of mitochondrial morphology in HSC and the metabolic control of HSC stemness by repressing Mfn2-mediated mitochondrial fusion.

Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

Characterization of hemocytes and hematopoietic cells of a freshwater crayfish based on single-cell transcriptome analysis

SummaryCrustaceans constitute a species-rich and ecologically important animal group, and their circulating blood cells (hemocytes) are of critical importance in immunity as key players in pathogen recognition, phagocytosis, melanization, and antimicrobial defense. To gain a better understanding of the immune responses to different pathogens, it is crucial that we identify different hemocyte subpopulations with different functions and gain a better understanding of how these cells are formed. Here, we performed single-cell RNA sequencing of isolated hematopoietic tissue (HPT) cells and hemocytes from the crayfish Pacifastacus leniusculus to identify hitherto undescribed hemocyte types in the circulation and show that the circulating cells are more diversified than previously recognized. In addition, we discovered cell populations in the HPT with clear precursor characteristics as well as cells involved in iron homeostasis, representing a previously undiscovered cell type. These findings may improve our understanding of hematopoietic stem cell regulation in crustaceans and other animals.

Early Growth Response Factor 1 in Aging Hematopoietic Stem Cells and Leukemia.

Aging is associated with various hematological disorders and a higher risk of myeloproliferative disorders. An aged hematopoietic system can be characterized by decreased immune function and increased myeloid cell production. Hematopoietic stem cells (HSCs) regulate the production of blood cells throughout life. The self-renewal and regenerative potential of HSCs determine the quality and quantity of the peripheral blood cells. External signals from the microenvironment under different conditions determine the fate of the HSCs to proliferate, self-renew, differentiate, or remain quiescent. HSCs respond impromptu to a vast array of extracellular signaling cascades such as cytokines, growth factors, or nutrients, which are crucial in the regulation of HSCs. Early growth response factor 1 (EGR1) is one of the key transcription factors controlling HSC proliferation and their localization in the bone marrow (BM) niche. Downregulation of Egr1 activates and recruits HSCs for their proliferation and differentiation to produce mature blood cells. Increased expression of Egr1 is implicated in immuno-aging of HSCs. However, dysregulation of Egr1 is associated with hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML). Here, we summarize the current understanding of the role of EGR1 in the regulation of HSC functionality and the manifestation of leukemia. We also discuss the alternative strategies to rejuvenate the aged HSCs by targeting EGR1 in different settings.

Metabolic Regulation of Hematopoietic Stem Cells.

Cellular metabolism is a key regulator of hematopoietic stem cell (HSC) maintenance. HSCs rely on anaerobic glycolysis for energy production to minimize the production of reactive oxygen species and shift toward mitochondrial oxidative phosphorylation upon differentiation. However, increasing evidence has shown that HSCs still maintain a certain level of mitochondrial activity in quiescence, and exhibit high mitochondrial membrane potential, which both support proper HSC function. Since glycolysis and the tricarboxylic acid (TCA) cycle are not directly connected in HSCs, other nutrient pathways, such as amino acid and fatty acid metabolism, generate acetyl-CoA and provide it to the TCA cycle. In this review, we discuss recent insights into the regulatory roles of cellular metabolism in HSCs. Understanding the metabolic requirements of healthy HSCs is of critical importance to the development of new therapies for hematological disorders.

Epigenetic modifier SMCHD1 maintains a normal pool of long-term hematopoietic stem cells

SummarySMCHD1 (structural maintenance of chromosomes hinge domain containing 1) is a noncanonical SMC protein that mediates long-range repressive chromatin structures. SMCHD1 is required for X chromosome inactivation in female cells and repression of imprinted and clustered autosomal genes, with SMCHD1 mutations linked to human diseases facioscapulohumeral muscular dystrophy (FSHD) and bosma arhinia and micropthalmia syndrome (BAMS). We used a conditional mouse model to investigate SMCHD1 in hematopoiesis. Smchd1-deleted mice maintained steady-state hematopoiesis despite showing an impaired reconstitution capacity in competitive bone marrow transplantations and age-related hematopoietic stem cell (HSC) loss. This phenotype was more pronounced in Smchd1-deleted females, which showed a loss of quiescent HSCs and fewer B cells. Gene expression profiling of Smchd1-deficient HSCs and B cells revealed known and cell-type-specific SMCHD1-sensitive genes and significant disruption to X-linked gene expression in female cells. These data show SMCHD1 is a regulator of HSCs whose effects are more profound in females.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

Megakaryocytes as the Regulator of the Hematopoietic Vascular Niche.

Megakaryocytes (MKs) are important components of the hematopoietic niche. Compared to the non-hematopoietic niche cells, MKs serving as part of the hematopoietic niche provides a mechanism for feedback regulation of hematopoietic stem cells (HSCs), in which HSC progeny (MKs) can modulate HSC adaptation to hematopoietic demands during both steady-state and stress hematopoiesis. MKs are often located adjacent to marrow sinusoids. Considering that most HSCs reside close to a marrow vascular sinusoid, as do MKs, the interactions between MKs and vascular endothelial cells are positioned to play important roles in modulating HSC function, and by extrapolation, might be dysregulated in various disease states. In this review, we discuss the interactions between MKs and the vascular niche in both normal and neoplastic hematopoiesis.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Abstract 3159: CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells

Abstract The mechanism by which stem and progenitor cells commit to proliferate, self-renew, or differentiate towards different lineages remains unclear. Understanding this process is essential to decipher both normal tissue development and aberrant differentiation associated with malignant transformation. CUX1 is a non-clustered HOX-family transcription factor that is recurrently mutated in hematopoietic and solid tumours and regulates cell fate in multiple developmental systems. We reported that CUX1 is required for normal hematopoietic homeostasis of both murine and human hematopoietic stem and progenitor cells (HSPCs). To date, the transcriptional role of CUX1 in HSPCs has been unclear, partly because of the complexity of the Cux1 locus. Namely, Cux1 shares exons with a distinct gene, Casp, which encodes a highly expressed golgi-associated protein. As most transcriptomic studies combine Casp and Cux1 transcripts, the nuances of Cux1 expression are concealed. To identify the transcriptional mechanism by which CUX1 regulates HSPC functions, we compared scRNA-seq data of Kit+lin- HSPCs from transgenic mice expressing inducible shRNAs targeting Cux1, Cux1mid and Cux1low, to control counterparts expressing an shRNA targeting renilla luciferase. We demonstrate that separating Cux1 from Casp transcripts provides more power to detect the influence of Cux1 in scRNA-seq data analysis. While cell numbers within most HSPC sub-populations are largely unchanged after five days of Cux1 knockdown, the transcriptomes within multiple clusters are disrupted. The effect size is greatest in Cux1low cells, compared to Cux1mid, consistent with dosage dependent roles of CUX1 in HSPCs. Across sub-populations, Cux1 levels correlated with gene signatures of “stemness,” and Cux1 knockdown disrupted this gene signature in the core hematopoietic stem cell (HSC) population. Concomitantly, low levels of Cux1 led to upregulation of cellular metabolism and G2/M cell cycle transition pathways in core HSCs, indicating that CUX1 regulates stem cell quiescence at the hematopoietic apex. We are currently integrating this data with epigenetic datasets to determine the mechanism by which CUX1 regulates critical hematopoietic transcriptional programs. Overall, understanding the regulatory function of CUX1 will provide insight into how HSCs commit to different fates and may ultimately lead to novel cancer therapeutic interventions. Citation Format: Weihan Liu, Stephanie Konecki, Tanner Martinez, Megan McNerney. CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3159.

Cell scientist to watch – Lena Ho

ABSTRACT Lena Ho studied biochemistry at the University of Wisconsin–Madison, USA, funded by a scholarship from the Singapore Economic Development Board. Lena then joined the lab of Gerald Crabtree at Stanford University for her PhD, where she investigated the function and mechanism of chromatin remodelling proteins in the epigenetic regulation of embryonic, haematopoietic and cancer stem cells. She then moved back to Singapore for a postdoc in Bruno Reversade's lab at the Institute of Medical Biology, A*STAR, where she found that some non-coding RNAs can in fact encode short peptides, exemplified by her discovery of the peptide hormone ELABELA. In 2017, Lena became Assistant Professor at Duke–NUS Medical School and the Institute of Medical Biology, A*STAR, where her Endogenous Peptides lab currently focuses on secreted and mitochondria-localised peptides to uncover new paradigms in cell biology and improve cardiometabolic and immune health.